Plant genetic engineering technology has made significant progress during the last decade. It has become possible to introduce stably foreign genes into plants. This has provided exciting opportunities for modern agriculture.
The use of chimeric selectable marker genes in plant cell transformation has considerably simplified the selection of transformed plant cells. For example, by the expression of such a marker gene, transformed plant cells can be made resistant to antibiotics that are cytotoxic or growth-suppressing to non-transformed cells. A commonly used chimeric marker gene contains the neomycin phosphotransferase-II or nptII coding region (Bevan et al (1983) Nature 304, 184-187: Fraley et al (1983) Proc. Natl. Acad. Sci USA 80, 4803-4807). The nptII gene confers resistance to kanamycin, neomycin and G-418 antibiotics on plant cells expressing the gene (Reynaerts et al (1987) Plant Mol. Biol. Manual, Gelvin, S. B. & Schilperoort, R. A. (eds), Kluwer, Dordrecht, sect. A9, pp. 1-16).
Chimeric marker genes have typically contained: a plant-expressible promoter (with a 5' untranslated region); DNA (such as the npt II gene) encoding a selectable marker; and a 3' end formation and polyadenylation region active in plants. Although the versatility of the nptII gene has been confirmed in chimeric marker genes in several plant systems over the years, there have been limitations on its use that have necessitated the development of alternative antibiotic-resistance genes for use in such chimeric selectable marker genes (Hayford et al (1988) Plant Physiol. 86, 1216). Furthermore, in many situations, a second complementary antibiotic-resistance gene has been needed for introduction into plants that have already been transformed with an antibiotic-resistance gene. Such alternative antibiotic-resistance genes already exist, but they often require the use of very toxic substrates and/or they do not allow efficient selection in all plant species. Certainly for species that are routinely vegetatively reproducible, like potato, antibiotic-resistance genes encoding different selectable markers, with different specific substrates, are required when different genes have to be engineered at different times into a plant.
Among the known antibiotic-resistance genes are those encoding aminoglycoside antibiotic-acetylating (AAC) enzymes, four types of which have been characterized (based on the position of the modified amino group of the 2-deoxystreptamine-derived aminoglycosides):AAC(1), AAC(2'), AAC(3) and AAC(6'). See Shaw et al (1989) Antimicrob. Agents & Chemotherapy 33, 2052-2062. High-pressure liquid chromatography (HPLC) analysis has demonstrated the differences among the acetylated products of these four types of enzymes, and aminoglycoside-resistance profiles can be used to identify the presence of each of these types of enzymes in a host strain (Shaw et al (1989) supra).
European patent publication ("EP") 0 289 478 (Rogers et al (1988), Hayford et al (1988) supra, and Carrer et al (1991) Plant Mol. biol. 17, 301-303 describe the selection on gentamycin of plants transformed with an aminoglycoside-3-N-acetyltransferase-encoding gene (the "aac(3) gene"). The aac(3)-IV gene was found to confer resistance to kanamycin (in Petunia), but the level of resistance was, at most, only sufficient for marginal selection (Hayford et al (1988) supra). These publications also describe supertransformation of tobacco, previously transformed with the nptII gene, with the aac(3) gene by selection on gentamycin-containing medium. EP 0 289 478 also describes the use of gentamycin as a substrate in the transformation of petunia, soybean, oilseed rape and alfalfa transformed with the aac(3) gene. Carrer et al (1991) supra also describes the transformation of tobacco plants with an aac(3)-I gene, only conferring resistance to gentamycin, whereby the gentamycin-resistant plants retain their sensitivity to kanamycin. According to Carrer et al (1991) supra, it may be more advantageous to use a selectable marker gene with a narrow substrate specificity in some cases.
The AAC(6')-encoding genes (the "aac(6') genes") constitute a class of different but related genes acetylating the 6' amino group of several aminoglycoside antibiotics. Several bacterial aac(6') genes have been cloned and sequenced. According to Davis (1986) In Antibiotics in Laboratory Medicine, pp. 474-489, (ed.) Lorian V., Williams & Wilkins, Baltimore, Md. and Phillips & Shannon (1984) British Med. Bull. 40, 28-35, AAC(6') acetylates tobramycin, kanamycin, amikacyn, neomycin, gentamycin C.sub.1A and C.sub.2, sissomycin and netilmycin, although with varying efficiencies depending on the kind of AAC(6'). Two subtypes of aac(6') genes have been characterized by their aminoglycoside resistance profiles: aac(6')-I genes and aac-(6') -II genes; the former subclass comprises the aac(6')-IV and -4 genes, and the latter subclass comprises the aac(6')-III gene (Shaw et al (1989) supra). However, other classifications of these genes have also been made.
Another acetyltransferase, phosphinotricin acetyltransferase, has also been found to be capable of conferring a selectable phenotype (i.e., a herbicide resistance) to plant cells (De Block et al (1987) EMBO J. 6, 2513-2518).
EP 0 248 207 (Wohlleben et al, 1987) describes a gentamycin-resistance gene that is active in Streptomyces and is obtainable from a strain of S. ghanaensis by total digestion with BglII.
French patent publication 2 601 965 (Courvalin, 1988) describes a bifunctional gene encoding AAC(6') and APH(2") activities, the cloning and sequencing of this gene, and the use of parts of the gene as a DNA probe for detecting antibiotic-resistance developement in bacterial cultures.